Western Blot Analysis of Hypoxia Signaling

Western blotting was performed as previously described [51 (link), 52 (link)]. Briefly, cells were collected and lysed in RAPI buffer. Cleared total cell lysate was denatured by boiling and loaded onto a 10% gradient SDS–PAGE. After electrophoretic separation, proteins were transferred to an Immobilon-P membrane. Membrane was blocked with SuperBlock Blocking Buffer, and probed with the primary antibody, anti-HIF-1α, anti-VEGF, anti-p-Akt, anti-β-actin (Santa Cruz, CA) and anti-p-ERK1/2 (Cell Signaling Technology, Vancouver, Canada), followed by incubation with a secondary antibody conjugated with horseradish peroxidase. The proteins of interest were detected by using SuperSignal West Pico Chemiluminescent Substrate kit.

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Gao J.L., Shui Y.M., Jiang W., Huang E.Y., Shou Q.Y., Ji X., He B.C., Lv G.Y, & He T.C. (2016). Hypoxia pathway and hypoxia-mediated extensive extramedullary hematopoiesis are involved in ursolic acid's anti-metastatic effect in 4T1 tumor bearing mice. Oncotarget, 7(44), 71802-71816.

Publication 2016

anti-HIF-1α anti-VEGF anti-p-Akt anti-β-actin anti-p-ERK1/2

Actin Antibody Buffer Cell Electrophoretic Erk1 Horseradish peroxidase Immobilon p Membrane Proteins Sds page Vegf

Corresponding Organization :

Other organizations : Zhejiang Chinese Medical University, University of Chicago Medical Center, Chongqing Medical University, Zhejiang University

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Variable analysis

independent variables

Not explicitly mentioned

dependent variables

Protein expression levels of HIF-1α, VEGF, p-Akt, and p-ERK1/2

control variables

Western blotting protocol previously described in references [51] and [52]
Cell lysis using RAPI buffer
Denaturation of total cell lysate by boiling
Separation of proteins by 10% gradient SDS–PAGE
Transfer of proteins to Immobilon-P membrane
Blocking of membrane with SuperBlock Blocking Buffer
Incubation with primary antibodies (anti-HIF-1α, anti-VEGF, anti-p-Akt, anti-β-actin, anti-p-ERK1/2)
Incubation with secondary antibody conjugated with horseradish peroxidase
Detection of proteins of interest using SuperSignal West Pico Chemiluminescent Substrate kit

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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